Pathway of Detergent‐Mediated and Peptide Ligand‐Mediated Refolding of Heterodimeric Class II Major Histocompatibility Complex (MHC) Molecules

Abstract

We investigated the mechanism of refolding and reassembly of recombinant a and p chains of the class 11 major histocompatibility molecules (MHC-11) HLA-DRB5*0101. Both chains were expressed in the cytosol of Esckerichin coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the a and p chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C, to C,, in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than shortchain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.Keywords: major histocompatibility complex; protein folding ; folding intermediate; heterodimer formation; detergent.In vitro and in living cells most proteins fold by passing a series of intermediates where at each stage an increase of structural organization is gained (Jaenicke, 1991 ;Matthews, 1993;Rudolph and Lilie, 1996). During a folding reaction hydrophobic regions are transiently exposed to water. This makes the intermediates susceptible to aggregation which is the dominant competing reaction of protein folding. In living cells aggregation is prevented by chaperone molecules (Ellis and Hartl, 1996). I n vitro, aggregation may be reduced by addition of cosolvents (Sawano et al., 1992;Cleland et al., 1992; Sirokmlin and Fasman, 1993;Buchner and Rudolph, 1991) MHC-IT. class-I1 major histocompatibility complex; HexOGlc, n-hexyl /I-i~-glucoside; OctOGlc, n-octyl 11-D-glucoside; DodOGlc, ii-dodecyl /j-u-glucaside; OctOGlc,, n-octyl /I-D-ghC0-4-(1-glucoside; DodOGlc2, n-dodecyl ~-u-gluco-4-u-glucoside; TetOGlc,, 12-tetradecyl /I-n-gluco-4-a-gluco...

External Factors Affecting Protein Aggregation

“…These include detergents and cyclodextrins, which are used to suppress formation of protein aggregates during protein refolding. 22,231,304,314,326,327 …”

Optimization of Inclusion Body Solubilization and Renaturation of Recombinant Human Growth Hormone from Escherichia coli

“…Chaotropic agents such as urea, guanidine hydrochloride (Gdn-HCl) 2 , and thiocyanate salts (8,9), detergents such as sodium dodecyl sulfate (SDS) (10), N-cetyltrimethylammonium chloride (11) and sarkosyl (sodium N-lauroyl sarcosine; NLS) (12) along with reducing agents like ␤-mercaptoethanol, dithiothreitol, or cysteine have been extensively used for solubilizing the inclusion body proteins. The soluble proteins are then refolded to their native state after the chaotropic agents or other salts are removed by dialyzing the proteins in buffers containing reducing and oxidizing agents (8,13).…”

Solubilization and Refolding of Inclusion Body Proteins

“…A major issue concerning these conventional solubilization agents is that they completely denature the solubilized protein molecules, which often aggregate again during refolding step. Chaotropic agents such as urea and GdnHCl, in the presence of low concentration of detergents such as sodium dodecyl sulfate (SDS) [ 6 ], sodium deoxycholate, and sodium N-lauroyl sarcosine [ 7 ] along with reducing agents like β-mercaptoethanol and dithiothreitol, have been extensively used for solubilizing the IB proteins. During the last years, there has been a great amount of research aiming to develop new strategies for solubilization of IBs.…”